Analysis of methylation using nucleic acid arrays

ABSTRACT

Arrays for genome-wide analysis of methylation are disclosed. IN a preferred aspect arrays comprising a plurality of probes complementary to a plurality of identified CpG islands in the human, mouse and rat genome are disclosed. The arrays may be used to detect methylation within cpG islands in samples from human, mouse and rat genomes.

RELATED APPLICATIONS

The present application is a divisional application of U.S. patentapplication Ser. No. 11/695,599, filed Apr. 2, 2007, now U.S. Pat. No.7,901,882, which claims priority to U.S. Provisional Application No.60/788,520, filed Mar. 31, 2006, the entire disclosures of which areincorporated by reference.

FIELD OF THE INVENTION

The present invention relates to arrays and methods for detectingmethylation of nucleic acids.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted on compact disk is hereby incorporated byreference. The file on the disk is named 3791.1sequencelist.txt, thefile is 108 MB and the date of creation is Mar. 30, 2007.

BACKGROUND OF THE INVENTION

The genomes of higher eukaryotes contain the modified nucleoside5-methyl cytosine (5-meC). This modification is usually found as part ofthe dinucleotide CpG. Cytosine is converted to 5-methylcytosine in areaction that involves flipping a target cytosine out of an intactdouble helix and transfer of a methyl group from S-adenosylmethionine bya methyltransferase enzyme (Klimasauskas et al., Cell 76:357-369, 1994).This enzymatic conversion is the only epigenetic modification of DNAknown to exist in vertebrates and is essential for normal embryonicdevelopment (Bird, Cell 70:5-8, 1992; Laird and Jaenisch, Human Mol.Genet. 3:1487-1495, 1994; and Li et al., Cell 69:915-926, 1992).

The frequency of the CpG dinucleotide in the human genome is only about20% of the statistically expected frequency, possibly because ofspontaneous deamination of 5-meC to T (Schoreret et al., Proc. Natl.Acad Sci. USA 89:957-961, 1992). There are about 28 million CpG doubletsin a haploid copy of the human genome and it is estimated that about70-80% of the cytosines at CpGs are methylated. Regions where CpG ispresent at levels that are approximately the expected frequency arereferred to as “CpG islands” (Bird, A. P., Nature 321:209-213, 1986).These regions have been estimated to comprise about 1% of vertebrategenomes and account for about 15% of the total number of CpGdinucleotides. CpG islands are typically between 0.2 and 1 kb in lengthand are often located upstream of housekeeping and tissue-specificgenes. CpG islands are often located upstream of transcribed regions,but may also extend into transcribed regions. About 2-4% of cytosinesare methylated and probably the majority of cytosines that are 5′ of Gsare methylated. Most of the randomly distributed CpGs are methylated,but only about 20% of the CpGs in CpG islands are methylated.

DNA methylation is an epigenetic determinant of gene expression.Patterns of CpG methylation are heritable, tissue specific, andcorrelate with gene expression. The consequence of methylation isusually gene silencing. DNA methylation also correlates with othercellular processes including embryonic development, chromatin structure,genomic imprinting, somatic X-chromosome inactivation in females,inhibition of transcription and transposition of foreign DNA and timingof DNA replication. When a gene is highly methylated it is less likelyto be expressed, possibly because CpG methylation prevents transcriptionfactors from recognizing their cognate binding sites. Proteins that bindmethylated DNA may also recruit histone deacetylase to condense adjacentchromatin. Such “closed” chromatin structures prevent binding oftranscription factors. Thus the identification of sites in the genomecontaining 5-meC is important in understanding cell-type specificprograms of gene expression and how gene expression profiles are alteredduring both normal development and diseases such as cancer. Precisemapping of DNA methylation patterns in CpG islands has become essentialfor understanding diverse biological processes such as the regulation ofimprinted genes, X chromosome inactivation, and tumor suppressor genesilencing in human cancer caused by increase methylation.

Methylation of cytosine residues in DNA plays an important role in generegulation. Methylation of cytosine may lead to decreased geneexpression by, for example, disruption of local chromatin structure,inhibition of transcription factor-DNA binding, or by recruitment ofproteins which interact specifically with methylated sequences andprevent transcription factor binding. DNA methylation is required fornormal embryonic development and changes in methylation are oftenassociated with disease. Genomic imprinting, X chromosome inactivation,chromatin modification, and silencing of endogenous retroviruses alldepend on establishing and maintaining proper methylation patterns.Abnormal methylation is a hallmark of cancer cells and silencing oftumor suppressor genes is thought to contribute to carcinogenesis.Methylation mapping using microarray-based approaches may be used, forexample, to profile cancer cells revealing a pattern of DNA methylationthat may be used, for example, to diagnose a malignancy, predicttreatment outcome or monitor progression of disease. Methylation ineukaryotes can also function to inhibit the activity of viruses andtransposons, see Jones et al., EMBO J 17:6385-6393 (1998). Alterationsin the normal methylation process have also been shown to be associatedwith genomic instability (Lengauer et al., Proc. Natl. Acad. Sci. USA94:2545-2550, 1997). Such abnormal epigenetic changes may be found inmany types of cancer and can serve as potential markers for oncogenictransformation.

SUMMARY OF THE INVENTION

The present invention provides methods and arrays for analysis ofmethylation status of sites in genomic DNA.

In a first aspect, the invention thus provides an array of more than250,000 different sequence probes that are complementary to regions ofthe human genome that are rich in CpG dinucleotides.

In one embodiment the array comprises probes to more than 25,000 CpGislands in the human genome. The islands are selected from annotatedislands in public databases, preferably the UCSC genome browser. Inpreferred aspects islands are selected that have a GC content of 50% orgreater over a length of 200 base pairs or greater.

In one embodiment the array comprises at least one probe set for eachCpG island to be interrogated and each probe set comprises 8, 20 to 30base probes that are perfectly complementary to a region of the CpGisland over the entire length of the probe.

In another aspect, the array comprises three probes sets comprising 8probes for each CpG island that is greater than 5 kb in length. Theprobe sets may be distributed so that the probes in one probe set arecomplementary to regions in the 5′ approximately 1,700 base pairs, onethe 3′ 1,700 base pairs and one the central 1,700 base pairs of theisland.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects and advantages of the present invention willbe apparent upon consideration of the following detailed descriptiontaken in conjunction with the accompanying drawings, in which likecharacters refer to like parts throughout, and in which:

FIG. 1 is a schematic illustrating a method of analyzing the methylationstatus of genomic DNA.

FIG. 2 is a schematic representation illustrating a method for enrichingfor DNA that is methylated.

FIG. 3A is an image of a gel showing the results of in vitro HpaIImethylation of Arabidopsis genomic clones followed by digestion withHpaII restriction enzyme. FIG. 3B is an image of a gel showing theresults of in vitro HpaII methylation of Arabidopsis genomic clonesfollowed by digestion with HpaII restriction enzyme.

FIG. 4 is a schematic of an experiment to detect methylated DNA enrichedaccording to the method illustrated in FIG. 2 using hybridization to anarray.

FIG. 5 is an image of a gel showing PCR amplicons of methylated andunmethylated control fragments amplified following digestion with HpaII.

DETAILED DESCRIPTION OF THE INVENTION a) General

The present invention has many preferred embodiments and relies on manypatents, applications and other references for details known to those ofthe art. Therefore, when a patent, application, or other reference iscited or repeated below, it should be understood that it is incorporatedby reference in its entirety for all purposes as well as for theproposition that is recited.

As used in this application, the singular form “a,” “an,” and “the”include plural references unless the context clearly dictates otherwise.For example, the term “an agent” includes a plurality of agents,including mixtures thereof.

An individual is not limited to a human being, but may also includeother organisms including but not limited to mammals, plants, fungi,bacteria or cells derived from any of the above.

Throughout this disclosure, various aspects of this invention can bepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

The practice of the present invention may employ, unless otherwiseindicated, conventional techniques and descriptions of organicchemistry, polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, and immunology, which arewithin the skill of the art. Such conventional techniques includepolymer array synthesis, hybridization, ligation, and detection ofhybridization using a label. Specific illustrations of suitabletechniques can be had by reference to the example herein below. However,other equivalent conventional procedures can, of course, also be used.Such conventional techniques and descriptions can be found in standardlaboratory manuals such as Genome Analysis: A Laboratory Manual Series(Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells: A LaboratoryManual, PCR Primer: A Laboratory Manual, and Molecular Cloning: ALaboratory Manual (all from Cold Spring Harbor Laboratory Press),Stryer, L. (1995) Biochemistry (4th Ed.) Freeman, New York, Gait,“Oligonucleotide Synthesis: A Practical Approach” 1984, IRL Press,London, Nelson and Cox (2000), Lehninger, Principles of Biochemistry3^(rd) Ed., W.H. Freeman Pub., New York, N.Y. and Berg et al. (2002)Biochemistry, 5^(th) Ed., W.H. Freeman Pub., New York, N.Y., all ofwhich are herein incorporated in their entirety by reference for allpurposes.

The present invention can employ solid substrates, including arrays insome preferred embodiments. Methods and techniques applicable to polymer(including protein) array synthesis have been described in U.S. Ser. No.09/536,841, WO 00/58516, U.S. Pat. Nos. 5,143,854, 5,242,974, 5,252,743,5,324,633, 5,384,261, 5,405,783, 5,424,186, 5,451,683, 5,482,867,5,491,074, 5,527,681, 5,550,215, 5,571,639, 5,578,832, 5,593,839,5,599,695, 5,624,711, 5,631,734, 5,795,716, 5,831,070, 5,837,832,5,856,101, 5,858,659, 5,936,324, 5,968,740, 5,974,164, 5,981,185,5,981,956, 6,025,601, 6,033,860, 6,040,193, 6,090,555, 6,136,269,6,269,846 and 6,428,752, in PCT Applications Nos. PCT/US99/00730(International Publication No. WO 99/36760) and PCT/US01/04285(International Publication No. WO 01/58593), which are all incorporatedherein by reference in their entirety for all purposes.

Patents that describe synthesis techniques in specific embodimentsinclude U.S. Pat. Nos. 5,412,087, 6,147,205, 6,262,216, 6,310,189,5,889,165, and 5,959,098. Nucleic acid arrays are described in many ofthe above patents, but the same techniques are applied to polypeptidearrays.

Nucleic acid arrays that are useful in the present invention includethose that are commercially available from Affymetrix (Santa Clara,Calif.) under the brand name GeneChip®. Example arrays are shown on thewebsite at affymetrix.com.

The present invention also contemplates many uses for polymers attachedto solid substrates. These uses include gene expression monitoring,profiling, library screening, genotyping and diagnostics. Geneexpression monitoring and profiling methods can be shown in U.S. Pat.Nos. 5,800,992, 6,013,449, 6,020,135, 6,033,860, 6,040,138, 6,177,248and 6,309,822. Genotyping and uses therefore are shown in U.S. Ser. Nos.10/442,021, 10/013,598 (U.S. Patent Application Publication20030036069), and U.S. Pat. Nos. 5,856,092, 6,300,063, 5,858,659,6,284,460, 6,361,947, 6,368,799 and 6,333,179. Other uses are embodiedin U.S. Pat. Nos. 5,871,928, 5,902,723, 6,045,996, 5,541,061, and6,197,506.

The present invention also contemplates sample preparation methods incertain preferred embodiments. Prior to or concurrent with hybridizationto an array, the sample may be amplified by a variety of mechanisms,some of which may employ PCR. See, for example, PCR Technology:Principles and Applications for DNA Amplification (Ed. H. A. Erlich,Freeman Press, NY, N.Y., 1992); PCR Protocols: A Guide to Methods andApplications (Eds. Innis, et al., Academic Press, San Diego, Calif.,1990); Mattila et al., Nucleic Acids Res. 19, 4967 (1991); Eckert etal., PCR Methods and Applications 1, 17 (1991); PCR (Eds. McPherson etal., IRL Press, Oxford); and U.S. Pat. Nos. 4,683,202, 4,683,195,4,800,159, 4,965,188, and 5,333,675. The sample may be amplified on thearray.

See, for example, U.S. Pat. No. 6,300,070 which is incorporated hereinby reference.

Other suitable amplification methods include the ligase chain reaction(LCR) (for example, Wu and Wallace, Genomics 4, 560 (1989), Landegren etal., Science 241, 1077 (1988) and Barringer et al. Gene 89:117 (1990)),transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA 86,1173 (1989) and WO88/10315), self-sustained sequence replication(Guatelli et al., Proc. Nat. Acad. Sci. USA, 87, 1874 (1990) andWO90/06995), selective amplification of target polynucleotide sequences(U.S. Pat. No. 6,410,276), consensus sequence primed polymerase chainreaction (CP-PCR) (U.S. Pat. No. 4,437,975), arbitrarily primedpolymerase chain reaction (AP-PCR) (U.S. Pat. Nos. 5,413,909,5,861,245), rolling circle amplification (RCA) (for example, Fire andXu, PNAS 92:4641 (1995) and Liu et al., J. Am. Chem. Soc. 118:1587(1996)) and nucleic acid based sequence amplification (NABSA), (See,U.S. Pat. Nos. 5,409,818, 5,554,517, and 6,063,603). Other amplificationmethods that may be used are described in, U.S. Pat. Nos. 5,242,794,5,494,810, 4,988,617 and in U.S. Ser. No. 09/854,317. Otheramplification methods are also disclosed in Dahl et al., Nuc. Acids Res.33(8):e71 (2005) and circle to circle amplification (C2CA) Dahl et al.,PNAS 101:4548 (2004). Locus specific amplification and representativegenome amplification methods may also be used.

Additional methods of sample preparation and techniques for reducing thecomplexity of a nucleic sample are described in Dong et al., GenomeResearch 11, 1418 (2001), in U.S. Pat. Nos. 6,872,529, 6,361,947,6,391,592 and 6,107,023, US Patent Publication Nos. 20030096235 and20030082543 and U.S. patent application Ser. No. 09/916,135.

Methods for conducting polynucleotide hybridization assays have beenwell developed in the art. Hybridization assay procedures and conditionswill vary depending on the application and are selected in accordancewith the general binding methods known including those referred to in:Maniatis et al. Molecular Cloning: A Laboratory Manual (2^(nd) Ed. ColdSpring Harbor, N.Y., 1989); Berger and Kimmel Methods in Enzymology,Vol. 152, Guide to Molecular Cloning Techniques (Academic Press, Inc.,San Diego, Calif., 1987); Young and Davism, P.N.A.S, 80: 1194 (1983).Methods and apparatus for carrying out repeated and controlledhybridization reactions have been described in U.S. Pat. Nos. 5,871,928,5,874,219, 6,045,996 and 6,386,749, 6,391,623 each of which areincorporated herein by reference.

The present invention also contemplates signal detection ofhybridization between ligands in certain preferred embodiments. See U.S.Pat. Nos. 5,143,854, 5,578,832; 5,631,734; 5,834,758; 5,936,324;5,981,956; 6,025,601; 6,141,096; 6,185,030; 6,201,639; 6,218,803; and6,225,625, in U.S. Ser. No. 10/389,194 and in PCT ApplicationPCT/US99/06097 (published as WO99/47964), each of which also is herebyincorporated by reference in its entirety for all purposes.

Methods and apparatus for signal detection and processing of intensitydata are disclosed in, for example, U.S. Pat. Nos. 5,143,854, 5,547,839,5,578,832, 5,631,734, 5,800,992, 5,834,758; 5,856,092, 5,902,723,5,936,324, 5,981,956, 6,025,601, 6,090,555, 6,141,096, 6,185,030,6,201,639; 6,218,803; and 6,225,625, in U.S. Ser. Nos. 10/389,194,60/493,495 and in PCT Application PCT/US99/06097 (published as WO99/47964), each of which also is hereby incorporated by reference in itsentirety for all purposes. Instruments and software may also bepurchased commercially from various sources, including Affymetrix.

The practice of the present invention may also employ conventionalbiology methods, software and systems. Computer software products of theinvention typically include computer readable medium havingcomputer-executable instructions for performing the logic steps of themethod of the invention. Suitable computer readable medium includefloppy disk, CD-ROM/DVD/DVD-ROM, hard-disk drive, flash memory, ROM/RAM,magnetic tapes and etc. The computer executable instructions may bewritten in a suitable computer language or combination of severallanguages. Basic computational biology methods are described in, forexample Setubal and Meidanis et al., Introduction to ComputationalBiology Methods (PWS Publishing Company, Boston, 1997); Salzberg,Searles, Kasif, (Ed.), Computational Methods in Molecular Biology,(Elsevier, Amsterdam, 1998); Rashidi and Buehler, Bioinformatics BasicsApplication in Biological Science and Medicine (CRC Press, London, 2000)and Ouelette and Bzevanis Bioinformatics: A Practical Guide for Analysisof Gene and Proteins (Wiley & Sons, Inc., 2^(nd) ed., 2001). See U.S.Pat. No. 6,420,108.

Methods for detection of methylation status are disclosed, for example,in Fraga and Esteller, BioTechniques 33:632-649 (2002) and Dahl andGuldberg Biogerontology 4:233-250 (2003). Methylation detection usingbisulfite modification and target specific PCR have been disclosed, forexample, in U.S. Pat. Nos. 5,786,146, 6,200,756, 6,143,504, 6,265,171,6,251,594, 6,331,393, and 6,596,493. U.S. Pat. No. 6,884,586 disclosedmethods for methylation analysis using nicking agents and isothermalamplification.

The present invention may also make use of various computer programproducts and software for a variety of purposes, such as probe design,management of data, analysis, and instrument operation. See, U.S. Pat.Nos. 5,593,839, 5,795,716, 5,733,729, 5,974,164, 6,066,454, 6,090,555,6,185,561, 6,188,783, 6,223,127, 6,229,911 and 6,308,170.

Additionally, the present invention may have preferred embodiments thatinclude methods for providing genetic information over networks such asthe Internet as shown in U.S. Ser. Nos. 10/197,621, 10/063,559 (UnitedStates Publication No. 20020183936), Ser. Nos. 10/065,856, 10/065,868,10/328,818, 10/328,872, 10/423,403, and 60/482,389.

All documents, i.e., publications and patent applications, cited in thisdisclosure, including the foregoing, are incorporated by referenceherein in their entireties for all purposes to the same extent as ifeach of the individual documents were specifically and individuallyindicated to be so incorporated by reference herein in its entirety.

b) Definitions

“Adaptor sequences” or “adaptors” or “linkers” are generallyoligonucleotides of at least 5, 10, or 15 bases and preferably no morethan 50 or 60 bases in length; however, they may be even longer, up to100 or 200 bases that may be attached to the ends of fragments. Adaptorsequences may be synthesized using any methods known to those of skillin the art. For the purposes of this invention they may, as options,comprise primer binding sites, recognition sites for endonucleases,common sequences and promoters. The adaptor may be entirely orsubstantially double stranded or entirely single stranded. A doublestranded adaptor may comprise two oligonucleotides that are at leastpartially complementary. The adaptor may be phosphorylated orunphosphorylated on one or both strands.

Adaptors may be ligated to blunt or sticky ends of fragments and theadaptors may have blunt or sticky ends. They may comprise asubstantially double stranded region and a short single stranded regionwhich is complementary to the single stranded region created bydigestion with a restriction enzyme, also referred to as a “sticky end.”Overhangs may also be converted to blunt ends by filling in an overhangor removing an overhang.

Methods of ligation will be known to those of skill in the art and aredescribed, for example in Sambrook et at. (2001) and the New EnglandBioLabs catalog. Methods include using T4 DNA Ligase which catalyzes theformation of a phosphodiester bond between juxtaposed 5′ phosphate and3′ hydroxyl termini in duplex DNA or RNA with blunt and sticky ends; TagDNA Ligase which catalyzes the formation of a phosphodiester bondbetween juxtaposed 5′ phosphate and 3′ hydroxyl termini of two adjacentoligonucleotides which are hybridized to a complementary target DNA; E.coli DNA ligase which catalyzes the formation of a phosphodiester bondbetween juxtaposed 5′-phosphate and 3′-hydroxyl termini in duplex DNAcontaining cohesive ends; and T4 RNA ligase which catalyzes ligation ofa 5′ phosphoryl-terminated nucleic acid donor to a 3′hydroxyl-terminated nucleic acid acceptor through the formation of a3′→5′ phosphodiester bond, substrates include single-stranded RNA andDNA as well as dinucleoside pyrophosphates; or any other methodsdescribed in the art.

Adaptors may also incorporate modified nucleotides that modify theproperties of the adaptor sequence. For example, phosphorothioate groupsmay be incorporated in one of the adaptor strands. A phosphorothioategroup is a modified phosphate group with one of the oxygen atomsreplaced by a sulfur atom. In a phosphorothioated oligo (often called an“S-Oligo”), some or all of the internucleotide phosphate groups arereplaced by phosphorothioate groups. The modified backbone of an S-Oligois resistant to the action of most exonucleases and endonucleases.Phosphorothioates may be incorporated between all residues of an adaptorstrand, or at specified locations within a sequence. A useful option isto sulfurize only the last few residues at each end of the oligo. Thisresults in an oligo that is resistant to exonucleases, but has a naturalDNA center.

The term “array” as used herein refers to an intentionally createdcollection of molecules which can be prepared either synthetically orbiosynthetically. The molecules in the array can be identical ordifferent from each other. The array can assume a variety of formats,for example, libraries of soluble molecules; libraries of compoundstethered to resin beads, silica chips, or other solid supports.

The term “combinatorial synthesis strategy” as used herein refers to acombinatorial synthesis strategy is an ordered strategy for parallelsynthesis of diverse polymer sequences by sequential addition ofreagents which may be represented by a reactant matrix and a switchmatrix, the product of which is a product matrix. A reactant matrix is al column by m row matrix of the building blocks to be added. The switchmatrix is all or a subset of the binary numbers, preferably ordered,between l and m arranged in columns. A “binary strategy” is one in whichat least two successive steps illuminate a portion, often half, of aregion of interest on the substrate. In a binary synthesis strategy, allpossible compounds which can be formed from an ordered set of reactantsare formed. In most preferred embodiments, binary synthesis refers to asynthesis strategy which also factors a previous addition step. Forexample, a strategy in which a switch matrix for a masking strategyhalves regions that were previously illuminated, illuminating about halfof the previously illuminated region and protecting the remaining half(while also protecting about half of previously protected regions andilluminating about half of previously protected regions). It will berecognized that binary rounds may be interspersed with non-binary roundsand that only a portion of a substrate may be subjected to a binaryscheme. A combinatorial “masking” strategy is a synthesis which useslight or other spatially selective deprotecting or activating agents toremove protecting groups from materials for addition of other materialssuch as amino acids.

The term “complementary” as used herein refers to the hybridization orbase pairing between nucleotides or nucleic acids, such as, forinstance, between the two strands of a double stranded DNA molecule orbetween an oligonucleotide primer and a primer binding site on a singlestranded nucleic acid to be sequenced or amplified. Complementarynucleotides are, generally, A and T (or A and U), or C and G. Two singlestranded RNA or DNA molecules are said to be complementary when thenucleotides of one strand, optimally aligned and compared and withappropriate nucleotide insertions or deletions, pair with at least about80% of the nucleotides of the other strand, usually at least about 90%to 95%, and more preferably from about 98 to 100%. Perfectlycomplementary refers to 100% complementarity over the length of asequence. For example, a 25 base probe is perfectly complementary to atarget when all 25 bases of the probe are complementary to a contiguous25 base sequence of the target with no mismatches between the probe andthe target over the length of the probe.

The term “CpG island” as used herein refers to stretches of DNA in agenome that are rich in GC relative to the rest of the genome. Typicallythe GC content is 50% or greater in these regions which extend overhundreds of base pairs and sometimes thousands. Often these regions markthe 5′ ends of genes.

The term “epigenetic” as used herein refers to factors other than theprimary sequence of the genome that affect the development or functionof an organism, they can affect the phenotype of an organism withoutchanging the genotype. Epigenetic factors include modifications in geneexpression that are controlled by heritable but potentially reversiblechanges in DNA methylation and chromatin structure. Methylation patternsare known to correlate with gene expression and in general highlymethylated sequences are poorly expressed.

The term “genome” as used herein is all the genetic material in thechromosomes of an organism. DNA derived from the genetic material in thechromosomes of a particular organism is genomic DNA. A genomic libraryis a collection of clones made from a set of randomly generatedoverlapping DNA fragments representing the entire genome of an organism.

The term “hybridization” as used herein refers to the process in whichtwo single-stranded polynucleotides bind non-covalently to form a stabledouble-stranded polynucleotide; triple-stranded hybridization is alsotheoretically possible. The resulting (usually) double-strandedpolynucleotide is a “hybrid.” Hybridizations are usually performed understringent conditions, for example, at a salt concentration of no morethan about 1 M and a temperature of at least 25° C. For example,conditions of 5×SSPE (750 mM NaCl, 50 mM NaPhosphate, 5 mM EDTA, pH 7.4)and a temperature of 25-30° C. are suitable for allele-specific probehybridizations or conditions of 100 mM MES, 1 M [Na⁺], 20 mM EDTA, 0.01%Tween-20 and a temperature of 30-50° C., preferably at about 45-50° C.Hybridizations may be performed in the presence of agents such asherring sperm DNA at about 0.1 mg/ml, acetylated BSA at about 0.5 mg/ml.As other factors may affect the stringency of hybridization, includingbase composition and length of the complementary strands, presence oforganic solvents and extent of base mismatching, the combination ofparameters is more important than the absolute measure of any one alone.Hybridization conditions suitable for microarrays are described in theGene Expression Technical Manual, 2004 and the GeneChip Mapping AssayManual, 2004, available at Affymetrix.com.

The term “hybridization probes” as used herein are oligonucleotidescapable of binding in a base-specific manner to a complementary strandof nucleic acid. Such probes include peptide nucleic acids, as describedin Nielsen et al., Science 254, 1497-1500 (1991), LNAs, as described inKoshkin et al. Tetrahedron 54:3607-3630, 1998, and U.S. Pat. No.6,268,490 and other nucleic acid analogs and nucleic acid mimetics.

The term “isolated nucleic acid” as used herein mean an object speciesinvention that is the predominant species present (i.e., on a molarbasis it is more abundant than any other individual species in thecomposition). Preferably, an isolated nucleic acid comprises at leastabout 50, 80 or 90% (on a molar basis) of all macromolecular speciespresent. Most preferably, the object species is purified to essentialhomogeneity (contaminant species cannot be detected in the compositionby conventional detection methods).

The term “label” as used herein refers to a luminescent label, a lightscattering label, a radioactive label or a molecule that interacts witha detectable label. Fluorescent labels include the commerciallyavailable fluorescein phosphoramidites such as Fluoreprime (Pharmacia),Fluoredite (Millipore) and FAM (ABI). See U.S. Pat. No. 6,287,778. Inpreferred aspects biotin is incorporated into nucleic acids and is usedto detectably label the nucleic acids through interaction withstreptavidin.

The term “mixed population” or sometimes refer by “complex population”as used herein refers to any sample containing both desired andundesired nucleic acids. As a non-limiting example, a complex populationof nucleic acids may be total genomic DNA, total genomic RNA or acombination thereof. Moreover, a complex population of nucleic acids mayhave been enriched for a given population but include other undesirablepopulations. For example, a complex population of nucleic acids may be asample which has been enriched for desired messenger RNA (mRNA)sequences but still includes some undesired ribosomal RNA sequences(rRNA).

The term “mRNA” or sometimes refer by “mRNA transcripts” as used herein,include, but not limited to pre-mRNA transcript(s), transcriptprocessing intermediates, mature mRNA(s) ready for translation andtranscripts of the gene or genes, or nucleic acids derived from the mRNAtranscript(s). Transcript processing may include splicing, editing anddegradation. As used herein, a nucleic acid derived from an mRNAtranscript refers to a nucleic acid for whose synthesis the mRNAtranscript or a subsequence thereof has ultimately served as a template.Thus, a cDNA reverse transcribed from an mRNA, an RNA transcribed fromthat cDNA, a DNA amplified from the cDNA, an RNA transcribed from theamplified DNA, etc., are all derived from the mRNA transcript anddetection of such derived products is indicative of the presence and/orabundance of the original transcript in a sample. Thus, mRNA derivedsamples include, but are not limited to, mRNA transcripts of the gene orgenes, cDNA reverse transcribed from the mRNA, cRNA transcribed from thecDNA, DNA amplified from the genes, RNA transcribed from amplified DNA,and the like.

The term “nucleic acid” as used herein refers to a polymeric form ofnucleotides of any length, either ribonucleotides, deoxyribonucleotidesor peptide nucleic acids (PNAs), that comprise purine and pyrimidinebases, or other natural, chemically or biochemically modified,non-natural, or derivatized nucleotide bases. The backbone of thepolynucleotide can comprise sugars and phosphate groups, as maytypically be found in RNA or DNA, or modified or substituted sugar orphosphate groups. A polynucleotide may comprise modified nucleotides,such as methylated nucleotides and nucleotide analogs. The sequence ofnucleotides may be interrupted by non-nucleotide components. Thus theterms nucleoside, nucleotide, deoxynucleoside and deoxynucleotidegenerally include analogs such as those described herein. These analogsare those molecules having some structural features in common with anaturally occurring nucleoside or nucleotide such that when incorporatedinto a nucleic acid or oligonucleoside sequence, they allowhybridization with a naturally occurring nucleic acid sequence insolution. Typically, these analogs are derived from naturally occurringnucleosides and nucleotides by replacing and/or modifying the base, theribose or the phosphodiester moiety. The changes can be tailor made tostabilize or destabilize hybrid formation or enhance the specificity ofhybridization with a complementary nucleic acid sequence as desired.

The term “primer” as used herein refers to a single-strandedoligonucleotide capable of acting as a point of initiation fortemplate-directed DNA synthesis under suitable conditions for example,buffer and temperature, in the presence of four different nucleosidetriphosphates and an agent for polymerization, such as, for example, DNAor RNA polymerase or reverse transcriptase. The length of the primer, inany given case, depends on, for example, the intended use of the primer,and generally ranges from 15 to 30 nucleotides. Short primer moleculesgenerally require cooler temperatures to form sufficiently stable hybridcomplexes with the template. A primer need not reflect the exactsequence of the template but must be sufficiently complementary tohybridize with such template. The primer site is the area of thetemplate to which a primer hybridizes. The primer pair is a set ofprimers including a 5′ upstream primer that hybridizes with the 5′ endof the sequence to be amplified and a 3′ downstream primer thathybridizes with the complement of the 3′ end of the sequence to beamplified.

The term “probe” as used herein refers to a surface-immobilized moleculethat can be recognized by a particular target. See U.S. Pat. No.6,582,908 for an example of arrays having all possible combinations ofprobes with 10, 12, and more bases. Examples of probes that can beinvestigated by this invention include, but are not restricted to,agonists and antagonists for cell membrane receptors, toxins and venoms,viral epitopes, hormones (for example, opioid peptides, steroids, etc.),hormone receptors, peptides, enzymes, enzyme substrates, cofactors,drugs, lectins, sugars, oligonucleotides, nucleic acids,oligosaccharides, proteins, and monoclonal antibodies.

Restriction enzymes or restriction endonucleases and their propertiesare well known in the art. A wide variety of restriction enzymes arecommercially available, from, for example, New England Biolabs.Restriction enzymes recognize a sequence specific sites (recognitionsite) in DNA. Typically the recognition site varies from enzyme toenzyme and may also vary in length. Isoschizomers are enzymes that sharethe same recognition site. Restriction enzymes may cleave close to orwithin their recognition site or outside of the recognition site. Oftenthe recognition site is symmetric because the enzyme binds the doublestranded DNA as homodimers. Recognition sequences may be continuous ormay be discontinuous, for example, two half sites separated by avariable region. Cleavage can generate blunt ends or short singlestranded overhangs.

In preferred aspects of the present invention enzymes that include atleast one CpG dinucleotide in the recognition site may be used. Enzymeswith a recognition site that includes the sequence CCGG include, forexample, Msp I, Hpa II, Age I, Xma I, Sma I, NgoM IV, Nae I, and BspE I.Enzymes with a recognition site that includes the sequence CGCG include,for example, BstU I, Mlu I, Sac II, BssH II and Nru I. Enzymes with arecognition site that includes the sequence GCGC include, for example,Hin P1 I, Hha I, Afe I, Kas I, Nar I, Sfo I, Bbe I, and Fsp I. Enzymeswith a recognition site that includes the sequence TCGA include, forexample, Taq I, Cla I, BspD I, PaeR7 I, Tli I, Xho I, Sal I, and BstB I.For additional enzymes that contain CpG in the recognition sequence.See, for example, the New England Biolabs catalog and web site. In someaspects two restriction enzymes may have a different recognitionsequence but generate identical overhangs or compatible cohesive ends.For example, the overhangs generated by cleavage with Hpa II or Msp Ican be ligated to the overhang generated by cleavage with Taq I. Somerestriction enzymes that include CpG in the recognition site are unableto cleave if the site is methylated, these are methylation sensitive.Other enzymes that contain CpG in their recognition site can cleaveregardless of the presence of methylation, these are methylationinsensitive. Examples of methylation insensitive enzymes, that include aCpG in the recognition site, include BsaW I (WCCGGW), BsoB I, BssS I,Msp I, and Taq I. Examples of methylation sensitive enzymes, thatinclude a CpG in the recognition site, include Aat II, Aci I, Acl I, AfeI, Age I, Asc I, Ava I, BmgB I, BsaA I, BsaH I, BspD I, Eag I, Fse I,Fau I, Hpa II, HinP1 I, Nar I, and SnaB I.

The term “solid support”, “support”, and “substrate” as used herein areused interchangeably and refer to a material or group of materialshaving a rigid or semi-rigid surface or surfaces. In many embodiments,at least one surface of the solid support will be substantially flat,although in some embodiments it may be desirable to physically separatesynthesis regions for different compounds with, for example, wells,raised regions, pins, etched trenches, or the like. According to otherembodiments, the solid support(s) will take the form of beads, resins,gels, microspheres, or other geometric configurations. See U.S. Pat. No.5,744,305 for exemplary substrates.

The term “target” as used herein refers to a molecule that has anaffinity for a given probe. Targets may be naturally-occurring orman-made molecules. Also, they can be employed in their unaltered stateor as aggregates with other species. Targets may be attached, covalentlyor noncovalently, to a binding member, either directly or via a specificbinding substance. Examples of targets which can be employed by thisinvention include, but are not restricted to, antibodies, cell membranereceptors, monoclonal antibodies and antisera reactive with specificantigenic determinants (such as on viruses, cells or other materials),drugs, oligonucleotides, nucleic acids, peptides, cofactors, lectins,sugars, polysaccharides, cells, cellular membranes, and organelles.Targets are sometimes referred to in the art as anti-probes. As the termtargets is used herein, no difference in meaning is intended. A “ProbeTarget Pair” is formed when two macromolecules have combined throughmolecular recognition to form a complex.

The term “tags” or “barcode tags” is used herein to refer to shortnucleic acid sequences of, for example, 15 to 50 bases, that may be usedto mark a molecule or mixture of interest. They are preferably part of aset of tags that function together as detectable markers that sharecommon hybridization properties and are not found in the molecule ofinterest. In preferred aspects tags are selected from random sequencesof the same length that are not found in a genome of interest, forexample, 20 mers not found in the human genome, and have similar meltingtemperatures from their respective tag probes which are the perfectcomplement of the tags. They may be designed algorithmically to maximizediscrimination on a microarray displaying complements of the respectivetags; a 1:1 correspondence as between tag sequence and nucleic acid towhich it is appended permits each such nucleic acid to be identified bydetection of the bar code uniquely associated therewith. See, e.g.,Shoemaker et al., Nature Genet. 14(4):450-6 (1996); EP 0799897; Fan etal., Genome Res. 10:853-60 (2000); and U.S. Pat. No. 6,150,516, thedisclosures of which are incorporated herein by reference in theirentireties.

CpG Island Arrays

Mammalian methylation patterns are complex and change duringdevelopment, see van Steensel and Henikoff BioTechniques 35: 346-357(2003). Methylation in promoter regions is generally accompanied by genesilencing and loss of methylation or loss of the proteins that bind tothe methylated CpG can lead to diseases in humans, for example,Immunodeficiency Craniofacial Syndrome and Rett Syndrome, Bestor (2000)Hum. Mol. Genet. 9:2395-2402. DNA methylation may be gene-specific andoccurs genome-wide.

Methods for detecting methylation status have been described in, forexample U.S. Pat. Nos. 6,214,556, 5,786,146, 6,017,704, 6,265,171,6,200,756, 6,251,594, 5,912,147, 6,331,393, 6,605,432, and 6,300,071 andUS Patent Application publication Nos. 20030148327, 20030148326,20030143606, 20030082609 and 20050009059, each of which are incorporatedherein by reference. Other array based methods of methylation analysisare disclosed in U.S. patent application Ser. No. 11/058,566 (Pg Pub20050196792 A1) and Ser. No. 11/213,273 (Pg Pub 20060292585 A1), whichare both incorporated herein by reference in their entireties. For areview of some methylation detection methods, see, Oakeley, E. J.,Pharmacology & Therapeutics 84:389-400 (1999). Available methodsinclude, but are not limited to: reverse-phase HPLC, thin-layerchromatography, SssI methyltransferases with incorporation of labeledmethyl groups, the chloracetaldehyde reaction, differentially sensitiverestriction enzymes, hydrazine or permanganate treatment (m5C is cleavedby permanganate treatment but not by hydrazine treatment), sodiumbisulfite, combined bisulphate-restriction analysis, and methylationsensitive single nucleotide primer extension.

Aberrant DNA methylation at CpG islands has been proven to play animportant role in the development of cancer. Genome-wide monitoring ofalterations in DNA methylation patterns will lead to new insights intothe early epigenetic events of tumor genesis and the discovery ofbiomarkers for cancer. CpG island microarrays to interrogate the vastmajority of CpG islands found in the genome and methods of using CpGisland microarrays are disclosed herein. The CpG island microarrays maybe used for rapid screening and high-throughput, genome-wide surveys ofDNA methylation status in, for example, mouse, human and rat. The arraydesign strategy, examples of detecting DNA methylation and data analysismethods are disclosed. A promoter tiling array may also be used as acomplementary method for methylation detection in human promoterregions. A promoter tiling array with over 25,000 promoters from humangenes is also discussed. The microarray technology disclosed herein maybe used, for example, for methylation research and biomarker discovery.

In a preferred aspect arrays containing probes that are selected tointerrogate regions that are candidates for methylation and forregulation by methylation are disclosed. Some embodiments are definedwith reference to the sequence listing that has been incorporated hereinby reference. The sequence listing contains the sequences of 743,256probes. Each sequence in the sequence listing represents the sequence ofa probe that may be present on a CpG island array. The sequences includea plurality of experimental probe sequences and a plurality of controlprobe sequences. The experimental probe sequences are SEQ ID NO.1-668,564 and the control sequences are SEQ ID NO. 668,565-743,256.Approximately 90% of the probes of the array are experimental probeswith approximately 10% of the probes as control probes.

The sequence listing contains experimental and control probes for human,mouse and rat arrays. The experimental human probes are SEQ ID NO.1-222,822. The experimental mouse probes are SEQ ID NO. 222,823-448,233.The experimental rat probes are SEQ ID NO. 448,234-668,564. The humancontrol probes are SEQ ID NO. 668,565-693,466. The mouse control probesare SEQ ID NO. 693,467-715,779. The rat control probes are SEQ ID NO.715,780-743,256.

In one aspect an array for analysis of methylation in humans isdisclosed. The array may comprise a plurality of experimental probesselected from the probes represented by SEQ ID NO. 1-222,822. The arraymay further comprise control probes selected from the control probesrepresented by SEQ ID NO. 668,565-693,466.

In another aspect an array for analysis of methylation in rat isdisclosed. The array may comprise a plurality of experimental probesselected from the probes represented by 448,234-668,564. The array mayfurther comprise control probes selected from the control probesrepresented by SEQ ID NO. 715,780-743,256

In another aspect an array for analysis of methylation in mouse isdisclosed. The array may comprise a plurality of experimental probesselected from the probes represented by SEQ ID NO. 222,823-448,233. Thearray may further comprise control probes selected from the controlprobes represented by SEQ ID NO. 693,467-715,779.

It should be understood that while the probes of an array may beselected to be perfectly complementary to the sequence of a selectedorganisms, for example, human, mouse or rat, that the array may beuseful for hybridization to related species that share sequencehomology. For example, an array designed to be complementary to humangenetic material may be used to analyze a sample from another primate,for example, gorilla.

In preferred aspects the arrays contain more than 200,000 probes thatare perfectly complementary to regions of the genome that are predictedto be CpG islands. The probes are preferably between 15 and 100 bases,more preferably between 20 and 50 bases and most preferably between 20and 30 bases. In one aspect more than 90% of the probes are 25 bases inlength. Probes are arranged on the array so that each different sequenceis present in a distinct feature or geographical location of the arrayand that location is known or determinable. Not all probes within afeature area need be full length.

In some aspects CpG island arrays may vary from other arrays in thefrequency of CG dinucleotides in the probes. Since the CG dinucleotideoccurs at a relatively low frequency, genome arrays such as expressionarrays or genotyping arrays typically have lower frequency of CG in theprobes. In one aspect more than 80, 85, 90 or 95% of the probes of thearray contain at least one CG dinucleotide. In preferred aspects morethan 50, 60, 70 or 80% of the probes of the array contain at least twoCG dinucleotides. A CG dinucleotide is a 5′-CG-3′ where the G and C arecontiguous in the nucleic acid chain and not separated by anothernucleotide. In one aspect at least 25% of the experimental probes haveat least 3 GC dinucleotides. In one aspect at least 7.5% of theexperimental probes of the array have 4 or more GC dinucleotides. Inpreferred aspects there are more than 100,000, 250,000, 500,000,750,000, 1,000,000, or 2,000,000 experimental probes on the array.

In one embodiment, probes are selected for the array so that for eachCpG island that is less than 5 kb the array includes a probe set of 8probes that target that island. For islands greater than 5 kb 3 probesets of 8 probes each were designed and included on the array. The 3probes sets were spaced roughly so that one interrogated the 3′ regionof the island, one the middle and one the 5′ region. If, for example,the island is of length 6 kb a first probes set may be designed to bases1-2000, a second to bases 2001-4000 and a third to bases 4001-6000 ofthe island.

In one embodiment all of the probes in a probe set are selected to bewithin the same 1600 bp or smaller region. This increases the likelihoodthat all probes in a probe set are on a common restriction fragment in amethylation assay.

In a preferred aspect, probe selection for islands that are greater than1 kb and less than 5 kb, begins in the central 1 kb of the island. Forislands greater than 5 kb, probe selection of a first probe set maystart within the central 1 kb of the fragments, the 3′-most 1 kb for asecond probe set and the 5′-most 1 kb for a third probe set.

In one embodiment the quality of a selected probe set may be evaluatedafter selection by assessing a probe set score. For those islands thathave a probe set score of less than 1.44 (probe set score is 0.18× thenumber of probes), the probe selection region may be extended by 150 bpat either end (300 bases overall) and probe selection is repeated. All_x sets were automatically failed and failed probe sets were not rescuebased on an average raw probe score. For regions that failed a secondround of probe selection the region may be further extended by 150 bp oneither end and probe selection can be repeated. In one embodiment probesets that are subjected to a third round of probe selection may beallowed to have fewer probes, for example, 5-8 probes per set.

In one embodiment, during probe selection, probes may be pruned versusthe repeat database ‘Repbase’, and pruned versus the islands themselves+/−500 bp of genomic sequence, and the Arabidopsis mRNA clones. Thiseliminates or minimizes crosshybridization to repeats, and reduces crosshybridization between islands, such that the signal detected should bespecific to a given island.

In a preferred embodiment the array is a 400 format array with featuresthat are about 5 microns by 5 microns. Increased or decreased array andfeature sizes may be used, for example, array format may be 49, 100, 700or 900 format and the feature size may be 1×1, 7×7, 11×11, or 18×18microns. Smaller and larger feature sizes or a mixture of the abovefeature sizes may also be used. In a preferred aspect the 400 formatarrays with 5 micron feature size have about 250,000 different features.Each feature may be a different probe. Some probes may be present inmore than a single feature. In a preferred aspect plus strand perfectmatch probes are used. Controls may be GC matched antigenomic controls(about 2600 probes at 100 probes per bin—each bin is a different GCcontent). Additional controls may include, for example, Arabidopsisgenomic controls and Pseudomonal genomic sequences. Controls may beselected to have GC content that is similar to the target sequences. Inone aspect probes to well characterized genes that are known to beassociated with cancer may be included on the array. In one aspectprobes to ten or more human genes that are known to be associated withcancer may be included on the array. Probes to repetitive sequence andto 18S and 28S ribosomal RNA genes may also be included.

In one aspect probe sets are included for genes that are known to beregulated by methylation. In one aspect a set of 15 genes was selected,and the orthologs of these from each species (when available) were tiledas 40-probe (perfect match-only) sets. Probes for 14 human genes (16 CpGislands), 13 mouse (13 CpG islands), and 11 rat genes (11 CpG islands),were tiled. The gene names and the location of CpG island associatedwith that gene, if any, are shown in table 1.

Human Mouse Rat CDKN2A Cdkn2a chr4: 88280326-88281746 Cdkn2a chr5:108916025-108916249 chr9: 21964579-21965306 CDKN2A Mgmt chr7:131261988-131262200 Mgmt chr1: 196779125-196779347 chr9:21984102-21985910 MGMT Mlh1 chrX: 87947039-87947267 Mlh1 chr8:115672549-115673779 chr10: 131154939-131155700 MLH1 chr3:37009233-37010360 Gstp1 chr19: 3826567-3826865 Gstp1 chr1:206783223-206783432 GSTP1 chr11: 67107505-67108529 Brca1 chr11:101372865-101373065 Brca1 chr10: 90586958-90587201 BRCA1 chr17:38531661-38531986 Cdh1 chr8: 105898932-105899662 Cdh1 chr19:36447500-36448076 CDH1 chr16: 67328536-67329845 Timp3 chr10:86260784-86261356 Timp3 chr7: 19736510-19737381 TIMP3 chr22:31521935-31522821 Dapk1 chr13: 59260184-59261075 Dapk1 chr17:9778756-9779643 DAPK1 chr9: 87342069-87343371 Rassf1 chr9:107619881-107620907 Rassf1 chr8: 112821629-112822624 RASSF1 chr3:50349269-50350633 Thbs1 No CpG Island Thbs1 No CpG island RASSF1 chr3:50352808-50353544 Rarb chr14: 15071137-15071693 Rarb chr15_random:195605-195857 THBS1 chr15: 37659820-37660859 Trp73 chr4:152632046-152632968 RARB No CpG Island Stk11 chr10: 80238679-80240278TP73 chr1: 3589603-3592793 Apc chr18: 34443822-34444584

In a preferred aspect the arrays are used to analyze a sample that hasbeen treated to differentiate between methylated and unmethylatedsequences. Methylation is an epigenetic modification of DNA andinformation about methylation is typically lost during most methods ofnucleic acid amplification such as PCR, random or semi-random primingbased amplification, or locus specific primer extension basedamplification. To identify regions that were methylated in a startingsample methods, such as the method shown in FIG. 1, may be used thatenrich for methylated sequences relative to unmethylated prior to orduring amplification, and those enriched sequences may be detected byhybridization to an array such as those arrays disclosed herein.

In FIG. 1 the genomic DNA 101 with methylation sites 103 marked by an(*) is fragmented in step 105, for example, by restriction digestion,shearing, nebulization, sonication, or endonuclease treatment. A mixture107 of methylated (marked with *) and unmethylated fragments isobtained. Aliquots of the mixture 107 may be treated differentially insteps 109 and 111 to enrich for methylated fragments in step 109 orunmethylated fragments in step 111. Step 109 generates a sample 113 thatis enriched for unmethylated fragments and step 111 generates a sample115 that is enriched for methylated fragments. The enriched fragmentsare amplified in steps 117 and 119 to generate amplified samples 121 and123. Amplification does not preserve methylation but amplificationproduct 121 is enriched for sequences that were not methylated in 101and amplification product 123 is enriched for sequences that weremethylated in 101. The enriched amplification products may be analyzedin steps 125 and 127 respectively by hybridization to arrays 129 and131. In a preferred aspect arrays 129 and 131 are arrays where more than50, 60, 70, 80 or 90% of the probes on the array are complementary to aregion that contains at least one CpG dinucleotide.

Alternatively methods that enrich for regions that are unmethylatedrelative to regions that are methylated may also be used. Methods forseparation of methylated from unmethylated nucleic acids have beendescribed, see, for example, US patent publication nos. 20010046669,20030157546, and 20030180775 which are each incorporated herein byreference in their entireties.

In a particularly preferred aspect, illustrated schematically in FIG. 2,a genomic sample 201 is fragmented in step 203 by a method that is notsensitive to methylation, HindIII digestion, for example. The fragments205 are ligated with adaptors in step 207, the adaptor ligated fragments209 are fragmented in step 211 using a method that is sensitive tomethylation, for example HpaII digestion. If the HpaII site ismethylated (indicated by an *) the site is resistant to cleavage byHpaII and fragment 209 remains intact. If the HpaII site is unmethylatedit is cleaved to form fragments 213 and 215 which have adaptors on onlyone end. In a subsequent amplification step 217 only the intact adaptorligated fragments are amplified to form amplicons 219 and the sample isamplified by PCR using primers to the adaptor sequences, see FIG. 2. Seealso, Huang et al., Hum. Mol. Genet. 8:459-470 (1999), Yan et al., Clin.Cancer Res. 6:1432-8 (2000) and U.S. Pat. No. 6,605,432. For anexplanation CpG islands and methods of analysis of methylation usingmicroarray methods see, Promoter and CpG Island Microarrays, Winegardenand Takashi (eds.) DNA Press (2005).

In some embodiments the methods include treatment of the sample withbisulfite. Unmethylated cytosine is converted to uracil through athree-step process during sodium bisulfite modification. The steps aresulphonation to convert cytosine to cytosine sulphonate, deamination toconvert cytosine sulphonate to uracil sulphonate and alkalidesulphonation to convert uracil sulphonate to uracil. Conversion onmethylated cytosine is much slower and is not observed at significantlevels in a 4-16 hour reaction. See Clark et al., Nucleic Acids Res.,22(15):2990-7 (1994). If the cytosine is methylated it will remain acytosine. If the cytosine is unmethylated it will be converted touracil. When the modified strand is copied, through, for example,extension of a locus specific primer, a random or degenerate primer or aprimer to an adaptor, a G will be incorporated in the interrogationposition (opposite the C being interrogated) if the C was methylated andan A will be incorporated in the interrogation position if the C wasunmethylated. When the double stranded extension product is amplifiedthose Cs that were converted to U's and resulted in incorporation of Ain the extended primer will be replaced by Ts during amplification.Those Cs that were not modified and resulted in the incorporation of Gwill remain as C.

Kits for DNA bisulfite modification are commercially available from, forexample, Human Genetic Signatures' Methyleasy and Chemicon's CpGenomeModification Kit. See also, WO04096825A1, which describes bisulfitemodification methods and Olek et al. Nuc. Acids Res. 24:5064-6 (1994),which discloses methods of performing bisulfite treatment and subsequentamplification on material embedded in agarose beads.

Bisulfite treatment allows the methylation status of cytosines to bedetected by a variety of methods. For example, any method that may beused to detect a SNP may be used, for examples, see Syvanen, Nature Rev.Gen. 2:930-942 (2001). Methods such as single base extension (SBE) maybe used or hybridization of sequence specific probes similar to allelespecific hybridization methods. In another aspect the MolecularInversion Probe (MIP) assay may be used.

The following Examples are offered by way of illustration only, and notby way of limitation.

Example 1

Human CpG island array. Sequences used in the design of the Human CpGIsland Array were selected from NCBI human genome assembly (Build 35,UCSC hg17, May 2004). Repetitive elements were removed by RepeatMasker.There are total of 27,801 CpG islands annotated by UCSC in NCBI humangenome assembly, as defined by an algorithm that measures expectedversus observed CpG composition, given the GC content of the segment.CpG islands were also filtered for GC content 50% or greater and lengthgreater than 200 bp. For a majority of the CpG islands, 8 probes wereselected as a probe set to represent each island. For the islands largerthan 5 kb, three probe sets (8 probes each) were selected.

The 27,801 islands represent an initial set of 27,771 that wasidentified in a first analysis. This first analysis missed 30 islands onrandom chr2 and random chr10. These 30 were subsequently processed andadded to the total to obtain 27,801. Of the initial 27,771 the sizedistribution was as follows: 5394 less than 300 bp, about 16,000 between300 and 1,000 bp, 5064 between 100 and 2000 bp, 1237 greater than 2 kband 177 greater than 5 kb. For the 177 islands that were greater than 5kb probe sets were designed and included on the array for each of the3′, middle and 5′ probe selection regions (PSRs) (3 distinct 8 probesets for each island).

For the 27,771 islands there were 28,125 PSRs (27771+177+177). 988 PSRsfailed round 1 and the failed PSRs were extended at either end by 150 bpand probe sets were re-selected in round 2. 261 PSRs failed round 2 andwere extended by a further 150 bp at either end and probe sets werere-selected in a round 3. 155 PSRs failed round 3. Of these 49 had probeset scores below a cutoff threshold and 106 had no probes selected atall. Of the 106 failed islands 62 were from 5 satellite repeate regionson chromosomes 4, 7(2) and 19(2). These satellites are not likely toplay a role in gene expression. The 30 islands from random chr2 andrandom chr10 all passed probe selection in the first round.

The 27,771 islands were analyzed by in silico restriction fragmentprediction. Exemplary results are shown in Table 2. Column 1 is sequenceid, column 2 is sequence length, column 3 is CpG island length, column 4is first position of cut site i, column 5 is sequence of cut site i,column 6 is first position of cutsite i+1, column 7 is sequence of cutsite i+1, column 8 is length of restriction fragment generated, column 9is number of CpG's with the fragment, column 10 indicates where the iand i+1 cut sites are relative to the cpg island: where “1” indicatesleft of the CpG island, “i” indicates internal (within the island) and“r” indicates right of the CpG island (the first letter denotes site iand the second letter denotes site i+1), column 11 denotes the probe setname and column 12 is the number of probes found entirely within thefragment (for the 27771 islands).

TABLE 2 1 2 3 4 5 6 7 8 9 10 11 12 chr10_0 2774 774 717 aatt 1954 aatt1258 78 lr CpG.1.1.S1 8 chr10_l 2552 552 835 aatt 1604 aatt 790 69 lrCpG.2.1.S1 8 chr10_10 2284 284 959 aatt 1533 aatt 595 35 lr CpG.3.1.S1 8chr10_100 2237 237 790 aatt 1395 aatt 626 23 lr CpG.4.1.S1 8 chr10_10003149 1149 593 aatt 1938 aatt 1366 137 li CpG.5.1.S1 6 chr10_1000 31491149 1959 aatt 2722 aatt 784 29 ir CpG.5.1.S1 2 chr10_1001 2249 249 247aatt 1395 aatt 1169 52 lr CpG.6.1.S1 8 chr10_1002 2414 414 104 aatt 1931aatt 1848 84 lr CpG.7.1.S1 8 chr10_1003 2461 461 839 aatt 2014 aatt 119662 lr CpG.8.1.S1 8 chr10_1004 2351 351 663 aatt 1409 aatt 767 44 lrCpG.9.1.S1 8 chr10_1005 2313 313 728 aatt 1344 aatt 637 50 lrCpG.10.1.S1 7 chr10_1005 2313 313 1383 aatt 1441 aatt 79 0 rrCpG.10.1.S1 1 chr10_1007 4587 2587 965 aatt 4382 aatt 3438 302 lrCpG.12.1.S1 8 chr10_1008 2300 300 485 aatt 2069 aatt 1605 73 lrCpG.13.1.S1 8 chr10_101 2231 231 944 aatt 1522 aatt 599 33 lrCpG.15.1.S1 8 chr10_1012 2221 221 1136 aatt 1452 aatt 337 18 irCpG.18.1.S1 4 chr10_1015 2928 928 825 aatt 1059 aatt 255 10 liCpG.21.1.S1 1 chr10_1015 2928 928 1080 aatt 2135 aatt 1076 102 irCpG.21.1.S1 7 chr10_1017 3688 1688 1191 aatt 2764 aatt 1594 229 irCpG.23.1.S1 8

Example 2

Human Promoter 1.0R array. Sequences used in the design of the HumanPromoter 1.0R Array were selected from NCBI human genome assembly (Build34). Repetitive elements were removed by RepeatMasker. Promoter regionswere selected using sequence information from 35,685 ENSEMBL genes(version 21_(—)34d May 14, 2004), 25,172 Refseq mRNAs (NCBI GenBank®Feb. 7, 2004), and 47,062 complete-CDS mRNA (NCBI GenBank® Dec. 15,2003). The probes selected for the Human Promoter 1.0R Array are asubset of the probes used in the Human Tiling 2.0R Array Set (P/N900772).

Oligonucleotide probes are synthesized in situ complementary to eachcorresponding sequence. Probes are tiled at an average resolution of 35bp, as measured from the central position of adjacent 25-mer probes,leaving a gap of approximately 10 bp between probes. Each promoterregion covers approximately 7.5 kb upstream through 2.45 kb downstreamof 5′ transcription start sites. For over 1,300 cancer-associated genes,coverage of promoter regions was expanded to include additional genomiccontent; for these selected genes total coverage spans from 10 kbupstream through 2.45 kb downstream of transcriptional start sites. Thearray interrogates regions proximal to transcription start sites andcontains probes for approximately 59% of CpG islands annotated by UCSCin NCBI human genome assembly (Build 34).

Example 3

To test the method of enrichment of methylated fragments in a sample anddetection of enrichment on an array a set of Arabidopsis genomiccontrols was used in combination with a CpG island array that containsprobes to the Arabidopsis controls. The general scheme is as shown inFIG. 2. Briefly, control DNAs that are either methylated or unmethylatedwere fragmented with HindIII and ligated to a HindIII adaptor sequence.The adaptor ligated fragments were then digested with HpaII which is amethylation sensitive restriction enzyme. Unmethylated fragments arefragmented while methylated fragments are not. Fragmentation of theunmethylated fragments blocks PCR amplification using a primer to theadaptor so only the fragments that were methylated at the HpaII site andwere not digested by HpaII will be amplified by PCR.

Seven different control DNAs, Ara1, Ara2, Ara6, Ara7, Arab, Ara9, andAra10 were used. The controls are each a different clone of anArabidopsis genomic DNA region cloned into plasmid pFC47 (Affymetrix).Inserts were confirmed by resequencing. The clones were eithermethylated by treatment with HpaII methylase (New England Biolabs) orare unmethylated. FIG. 3 shows confirmation of methylation by digestionof the methylated (odd numbered lanes) and unmethylated (even numberedlanes) controls with HpaII. As expected the methylated fragments (lanes1, 3, 5, 7, 9, and 11 in 3A and 1, 3, 5, and 7 in 3B) are resistant tofragmentation by HpaII and the unmethylated fragments (lanes 2, 4, 6, 8,10 and 12 in 3A and 2, 4, 6 and 8 in 3B) are not.

Enrichment of methylated fragments in spiked samples. FIG. 4 shows theexperimental design. Samples 1 and 2 are mixtures of the 7 control DNAsdiscussed above. In Sample 1 DNAs Ara 1, 2 and 6 are unmethylated andAra7, 8, 9 and 10 are methylated using HpaII methylase. In Sample 2 DNAsAra1, 2 and 6 are methylated using HpaII methylase and Ara7, 8, 9 and 10are unmethylated. The fragments are digested with HpaII restrictionenzyme in step 401 and subjected to PCR amplification in step 403 usingcommon flanking priming sites present in pFC47, and common primers “458”and “428”. The PCR amplicons were mixed with a complex background andthe mixture was fragmented and labeled in step 405, hybridized to anarray in step 407 and analyzed in step 409. The results for sample 2were then compared to the results from sample 1.

An aliquot of the PCR amplicons for Ara2, 3 and 9 were separated on agel shown in FIG. 5. Lane L is a ladder, lanes 1 and 2 are methylatedand unmethylated Ara2, respectively, lanes 3 and 4 are methylated andunmethylated Ara3 and lanes 11 and 12 are methylated and unmethylatedAra9. The position of the HpaII containing fragment amplicon (501, 503,505 and 507) are marked by arrows. The gel shows that after HpaIIdigestion the fragments are observed only in the methylated samples,lanes 1, 3 and 11 and not in the unmethylated samples, lanes 2, 4 and12.

The PCR amplicons were mixed with 18 micrograms of human genomic DNA,fragmented and end labeled using DLR and TdT. Triplicate samples weregenerated. Approximately 1 pM of each methylated or unmethylated Aragenomic clone was used in a final 80 μl hybridization solution. Thelabeled, fragmented samples were hybridized to the CpG island array andthe hybridization pattern was analyzed to compare Sample 1 to Sample 2.The arrays were hybridized in 1×MES, 0.7 M NaCl, 20 mM EDTA, 0.01% Tween20, 2.5×Denhart's solution, 20 μg Cot DNA, 10% DMSO, and 40 μM oligo B2at 45° C. for 16 hours. The arrays were washed with Affymetrix fluidicsstation using protocol FS450-0003 and scanned using the AffymetrixGCS3000. Cel files were generated using GCOS. Tiling Analysis Software(TAS) was used to obtain the signal intensities and p-value of pair wisecomparison. Integrated Genome Browser (IGB) was applied to display andview differential signal intensities of the probes with genome locationannotation.

As expected enrichment of HpaII containing fragments of Ara7, 8, 9 and10 was observed in Sample 1 relative to Sample 2 and enrichment of HpaIIcontaining fragments of Ara1, 2 and 6 was observed in Sample 2 relativeto Sample 1. Methylated fragments were enriched relative to unmethylatedfragments.

Example 4

Mouse CpG island array. For the mouse array the data source was UCSC mm6(March 2005). Initially 16,100 islands were identified, 5 of which weregreater than or equal to 5 kb, for each of these 5 islands 3 probe sets(3′, middle, and 5′ PSRs) were designed and included on the array. Forthe other 16,095 islands a single probe set was included on the array.The total number of island PSRs used for probe selection in the firstround was 16,110 (16,100+5+5).

During probe selection, the information was hard pruned versus therepeat database ‘Repbase’, and soft-pruned versus the islands themselves+/−500 bp of genomic sequence to eliminate cross-hybridization torepeats, and to reduce cross-hybridization between islands so that thesignal detected is specific to a given island.

414 PSRs failed round 1, and were extended by 150 bp at either end, andsubjected to reselection of probe sets in a round 2 of probe selection.98 PSRs failed round 2, and were subsequently extended by a further 150bp at each end and subject to a 3^(rd) round of probe selection. 56 PSRsfailed round 3. Of these, 26 had probe set scores below a thresholdcutoff, and 30 had no probes picked at all. The 26 were included in thefinal array design, but the 30 were not. The array design resulted in223,689 probes.

Example 5

Rat CpG island array. Probe selection started using 15832 islands, ofwhich 10 were greater than or equal to 5 kb. The 10 large islands wereeach divided into 3 regions for probe selection designated 3′, Middleand 5′ PSRs. In all there were 15,852 PSRs in the first round(15832+10+10).

During probe selection, the sequence were hard pruned versus the repeatdatabase ‘Repbase’, and soft-pruned versus the islands themselves +/−500bp of genomic sequence. The intent was to eliminate cross-hybridizationto repeats, and to reduce cross-hybridization between islands, such thatthe signal detected would be specific to a given island, as discussedabove.

535 PSRs failed round 1, and were extended by 150 bp at either end, andprobe sets were re-selected in a round 2. 269 PSRs failed round 2, andwere extended by a further 150 bp at either end, and probe sets werere-selected in a round 3. 189 PSRs failed round 3. Of these, 55 hadprobe set scores below a threshold cutoff, and 134 had no probes pickedat all. The 55 were included in the final array design, but the 134 werenot. The rat array design resulted in 218,637 probes

CONCLUSION

All patents, patent publications, and other published referencesmentioned herein are hereby incorporated by reference in theirentireties as if each had been individually and specificallyincorporated by reference herein. While preferred illustrativeembodiments of the present invention are described, one skilled in theart will appreciate that the present invention may be practiced by otherthan the described embodiments, which are presented for purposes ofillustration only and not by way of limitation. The present invention islimited only by the claims that follow.

What is claimed is:
 1. A method of selecting probes to be included in anarray of probes comprising: identifying a plurality of CpG islandswherein said plurality comprises more than 10,000 different CpG islandsin the genome of a single organism; defining the ends of each CpG islandand thereby identifying a probe selection region for each CpG island inthe plurality; selecting a probe set for each probe selection region,wherein the probes in each selected probe set are perfectlycomplementary to the probe selection region targeted by that probe set;assessing a probe set score for each selected probe set, wherein theprobe set score equals 0.18× the number of probes in the set; and foreach selected probe set having a probe set score of less than 1.44,extending the probe selection region by 150 bps at either end and thenselecting a probe set from the extended probe selection region, whereineach of the steps of identifying, defining, and selecting is performedby a computer, and wherein the computer that performs the identifyingstep is programmed to define a CpG island as a genomic region that is atleast 200 bases in length and has a GC content of at least 50%.
 2. Themethod of claim 1, further comprising: assessing a probe set score foreach probe set selected from an extended probe selection region; and foreach probe set selected from an extended probe selection region whichhas a probe set score of less than 1.44, extending the extended probeselection region by another 150 bps at either end and then selecting aprobe set from the twice-extended probe selection region.
 3. The methodof claim 1 further comprising identifying a plurality of genes in saidorganism that are regulated by the methylation and selecting a probe setfor each of said genes.
 4. The method of claim 1 wherein the organism ishuman, mouse, or rat.
 5. The method of claim 1, wherein each probe sethas at least 5 to 8 probes.
 6. The method of claim 1, wherein the probesin a probe set are selected to be within a region of 1600 bp or less. 7.The method of claim 1, further comprising pruning the selected probesagainst a repeat database and/or the plurality of identified CpG islands+/−500 bp of genomic sequence.
 8. The method of claim 1, furthercomprising synthesizing the probe sets selected from the probe selectionand extended probe selection regions.